Identification of a conserved 5'-dRP lyase activity in bacterial DNA repair ligase D and its potential role in base excision repair.

Ana De Ory,Marina Raguse,Olga Zafra,Ralf Moeller,Begoña Carrasco,Katja Nagler,Miguel De Vega

doi:10.1093/nar/gkw054

Abstract

Bacillus subtilis is one of the bacterial members provided with a nonhomologous end joining (NHEJ) system constituted by the DNA-binding Ku homodimer that recruits the ATP-dependent DNA Ligase D (BsuLigD) to the double-stranded DNA breaks (DSBs) ends. BsuLigD has inherent polymerization and ligase activities that allow it to fill the short gaps that can arise after realignment of the broken ends and to seal the resulting nicks, contributing to genome stability during the stationary phase and germination of spores. Here we show that BsuLigD also has an intrinsic 5′-2-deoxyribose-5-phosphate (dRP) lyase activity located at the N-terminal ligase domain that in coordination with the polymerization and ligase activities allows efficient repairing of 2′-deoxyuridine-containing DNA in an in vitro reconstituted Base Excision Repair (BER) reaction. The requirement of a polymerization, a dRP removal and a final sealing step in BER, together with the joint participation of BsuLigD with the spore specific AP endonuclease in conferring spore resistance to ultrahigh vacuum desiccation suggest that BsuLigD could actively participate in this pathway. We demonstrate the presence of the dRP lyase activity also in the homolog protein from the distantly related bacterium Pseudomonas aeruginosa, allowing us to expand our results to other bacterial LigDs.

Highlights

DNA double strand breaks (DSBs) are the most dangerous lesions whose repair is essential for maintenance of genome integrity [1]
Filling with CTP allowed final ligation of the nick (49% of the 19-mer 5 -P molecules) to yield a repaired 35-mer long molecule, reflecting a strong propensity of the enzyme for sealing nicks with a monoribonucleotide on the 3 end of the break, a functional signature of bacterial nonhomologous end joining (NHEJ) ligases that distinguishes them from the other polynucleotide ligases [47,48]
The absence of ExoA and/or Nfo in deletion mutant strains of B. subtilis sensitized the spores to treatments that damage spore DNA through generation of AP sites and strand breaks, suggesting that Base Excision Repair (BER) should be active to repair the lesions during spore germination and outgrowth that have accumulated during spore dormancy [43,44,45]

Summary

Introduction

DNA double strand breaks (DSBs) are the most dangerous lesions whose repair is essential for maintenance of genome integrity [1]. Many bacterial species spend much of their life cycle in stationary phase during which only a single copy of the chromosome is present In most of these cases the bacterium is endowed with a two-component nonhomologous end-joining (NHEJ) system [3] that mends DSBs through the direct joining of the DNA ends [3,4]. Bacterial NHEJ is composed of the homodimer Ku, homologous to the eukaryotic counterpart [5,6], and the dedicated multifunctional ATP-dependent DNA ligase D (LigD). Extensive characterization of these proteins both in vitro and in vivo has allowed envisioning how NHEJ operates in bacteria [7,8,9,10]. Due to the processing of the ends by nucleolytic and/or polymerization activities before final ligation, this pathway is often mutagenic [8,13,14]

Methods

Results

Conclusion